RESUMEN
Although chimeric antigen receptor (CAR) T cells have emerged as highly effective treatments for patients with hematologic malignancies, similar efficacy has not been achieved in the context of solid tumors. There are several reasons for this disparity including a) fewer solid tumor target antigens, b) heterogenous target expression amongst tumor cells, c) poor trafficking of CAR T cells to the solid tumor and d) an immunosuppressive tumor microenvironment (TME). Oncolytic viruses have the potential to change this paradigm by a) directly lysing tumor cells and releasing tumor neoantigens, b) stimulating the local host innate immune response to release cytokines and recruit additional innate and adaptive immune cells, c) carrying virus-encoded transgenes to "re-program" the TME to a pro-inflammatory environment and d) promoting an adaptive immune response to the neoantigens in this newly permissive TME. Here we show that the Tumor-Specific Immuno-Gene (T-SIGn) virus NG-347 which encodes IFNα, MIP1α and CD80 synergizes with anti-EGFR CAR T cells as well as anti-HER-2 CAR T cells to clear A549 human tumor xenografts and their pulmonary metastases at doses which are subtherapeutic when each is used as a sole treatment. We show that NG-347 changes the TME to a pro-inflammatory environment resulting in the recruitment and activation of both CAR T cells and mouse innate immune cells. We also show that the transgenes encoded by the virus are critical as synergy is lost in their absence.
Asunto(s)
Neoplasias Pulmonares , Receptores Quiméricos de Antígenos , Animales , Antígenos de Neoplasias/genética , Xenoinjertos , Humanos , Inmunoterapia Adoptiva/métodos , Neoplasias Pulmonares/terapia , Ratones , Receptores Quiméricos de Antígenos/genética , Linfocitos T , Microambiente TumoralRESUMEN
BACKGROUND: Treatment outcomes remain poor in recurrent platinum-resistant ovarian cancer. Enadenotucirev, a tumor-selective and blood stable adenoviral vector, has demonstrated a manageable safety profile in phase 1 studies in epithelial solid tumors. METHODS: We conducted a multicenter, open-label, phase 1 dose-escalation and dose-expansion study (OCTAVE) to assess enadenotucirev plus paclitaxel in patients with platinum-resistant epithelial ovarian cancer. During phase 1a, the maximum tolerated dose of intraperitoneally administered enadenotucirev monotherapy (three doses; days 1, 8 and 15) was assessed using a 3+3 dose-escalation model. Phase 1b included a dose-escalation and an intravenous dosing dose-expansion phase assessing enadenotucirev plus paclitaxel. For phase 1a/b, the primary objective was to determine the maximum tolerated dose of enadenotucirev (with paclitaxel in phase 1b). In the dose-expansion phase, the primary endpoint was progression-free survival (PFS). Additional endpoints included response rate and T-cell infiltration. RESULTS: Overall, 38 heavily pretreated patients were enrolled and treated. No dose-limiting toxicities were observed at any doses. However, frequent catheter complications led to the discontinuation of intraperitoneal dosing during phase 1b. Intravenous enadenotucirev (1×1012 viral particles; days 1, 3 and 5 every 28-days for two cycles) plus paclitaxel (80 mg/m2; days 9, 16 and 23 of each cycle) was thus selected for dose-expansion. Overall, 24/38 (63%) patients experienced at least 1 Grade ≥3 treatment-emergent adverse event (TEAE); most frequently neutropenia (21%). Six patients discontinued treatment due to TEAEs, including one patient due to a grade 2 treatment-emergent serious AE of catheter site infection (intraperitoneal enadenotucirev monotherapy). Among the 20 patients who received intravenous enadenotucirev plus paclitaxel, 4-month PFS rate was 64% (median 6.2 months), objective response rate was 10%, 35% of patients achieved stable disease and 65% of patients had a reduction in target lesion burden at ≥1 time point. Five out of six patients with matched pre-treatment and post-treatment biopsies treated with intravenous enadenotucirev plus paclitaxel had increased (mean 3.1-fold) infiltration of CD8 +T cells in post-treatment biopsies. CONCLUSIONS: Intravenously dosed enadenotucirev plus paclitaxel demonstrated manageable tolerability, an encouraging median PFS and increased tumor immune-cell infiltration in platinum-resistant ovarian cancer. TRIAL REGISTRATION NUMBER: NCT02028117.
Asunto(s)
Adenoviridae/genética , Carcinoma Epitelial de Ovario/terapia , Resistencia a Antineoplásicos , Neoplasias Ováricas/terapia , Paclitaxel/uso terapéutico , Platino (Metal)/farmacología , Adulto , Anciano , Animales , Antineoplásicos Fitogénicos/uso terapéutico , Carcinoma Epitelial de Ovario/genética , Carcinoma Epitelial de Ovario/patología , Terapia Combinada , Evaluación Preclínica de Medicamentos , Femenino , Estudios de Seguimiento , Humanos , Masculino , Dosis Máxima Tolerada , Ratones , Persona de Mediana Edad , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Pronóstico , Tasa de SupervivenciaRESUMEN
Ionising radiation causes cell death through the induction of DNA damage, particularly double-stranded DNA (dsDNA) breaks. Evidence suggests that adenoviruses inhibit proteins involved in the DNA damage response (DDR) to prevent recognition of double-stranded viral DNA genomes as cellular dsDNA breaks. We hypothesise that combining adenovirus treatment with radiotherapy has the potential for enhancing tumour-specific cytotoxicity through inhibition of the DDR and augmentation of virus production. We show that EnAd, an Ad3/Ad11p chimeric oncolytic adenovirus currently being trialled in colorectal and other cancers, targets the DDR pathway at a number of junctures. Infection is associated with a decrease in irradiation-induced 53BP1 and Rad51 foci formation, and in total DNA ligase IV levels. We also demonstrate a radiation-associated increase in EnAd production in vitro and in a pilot in vivo experiment. Given the current limitations of in vitro techniques in assessing for synergy between these treatments, we adapted the plaque assay to allow monitoring of viral plaque size and growth and utilised the xCELLigence cell adhesion assay to measure cytotoxicity. Our study provides further evidence on the interaction between adenovirus and radiation in vitro and in vivo and suggests these have at least an additive, and possibly a synergistic, impact on cytotoxicity.
RESUMEN
BACKGROUND: Enadenotucirev is a chimeric adenovirus with demonstrated preclinical tumor-selective cytotoxicity and a short half-life. Further clinical mechanism of action data showed that enadenotucirev can gain access to and replicate within different types of epithelial tumors. This phase 1 dose escalation study assessed intravenous (IV) dose escalation with enadenotucirev to establish the maximum tolerated dose (MTD) and subsequently identify a suitable schedule for repeated cycles. METHODS: Sixty-one patients with advanced epithelial tumors unresponsive to conventional therapy were enrolled and received enadenotucirev monotherapy as part of this study. During the phase 1a dose escalation (n = 22) and expansion (n = 9), delivery of enadenotucirev between 1 × 1010 and 1 × 1013 viral particles (vp) on days 1, 3, and 5 (single cycle) was used to determine an appropriate MTD. Subsequent treatment cohorts (phase 1a, n = 6 and phase 1b, n = 24) examined the feasibility of repeated dosing cycles in either 3-weekly or weekly dosing regimens. RESULTS: Enadenotucirev displayed a predictable and manageable safety profile at doses up to the MTD of 3 × 1012 vp, irrespective of infusion time or dosing schedule. The most commonly reported treatment-emergent adverse events (TEAEs) of grade 3 or higher were hypoxia, lymphopenia, and neutropenia. The frequency of all TEAEs (notably pyrexia and chills) was highest within 24 h of the first enadenotucirev infusion and decreased upon subsequent dosing. Additionally, delivery of three doses of enadenotucirev over 5 days optimized pharmacokinetic and chemokine profiles in the circulation over time. CONCLUSIONS: This study provides key clinical data in patients with solid epithelial tumors following treatment with IV enadenotucirev monotherapy and supports further investigation of enadenotucirev in combination with other therapeutic agents at doses up to the MTD of 3 × 1012 vp. TRIAL REGISTRATION: ( ClinicalTrials.gov Identifier: NCT02028442 ). Trial registration date: 07 January 2014 - Retrospectively registered.
Asunto(s)
Adenoviridae , Neoplasias Glandulares y Epiteliales/terapia , Viroterapia Oncolítica , Virus Oncolíticos , Administración Intravenosa , Adulto , Anciano , Citocinas/sangre , Femenino , Humanos , Masculino , Dosis Máxima Tolerada , Persona de Mediana Edad , Neoplasias Glandulares y Epiteliales/sangre , Neoplasias Glandulares y Epiteliales/virologíaRESUMEN
Oncolytic viruses represent an emerging approach to cancer therapy. However, better understanding of their interaction with the host cancer cell and approaches to enhance their efficacy are needed. Here, we investigate the effect of chemically induced endoplasmic reticulum (ER) stress on the activity of the chimeric group B adenovirus Enadenotucirev, its closely related parental virus Ad11p, and the archetypal group C oncolytic adenovirus Ad5. We show that treatment of colorectal and ovarian cancer cell lines with thapsigargin or ionomycin caused an influx of Ca2+, leading to an upregulation in E1A transcript and protein levels. Increased E1A protein levels, in turn, increased levels of expression of the E2B viral DNA polymerase, genome replication, late viral protein expression, infectious virus particle production, and cell killing during Enadenotucirev and Ad11p, but not Ad5, infection. This effect was not due to the induction of ER stress, but rather the influx of extracellular Ca2+ and consequent increase in protein kinase C activity. These results underscore the importance of Ca2+ homeostasis during adenoviral infection, indicate a signaling pathway between protein kinase C and E1A, and raise the possibility of using Ca2+ flux-modulating agents in the manufacture and potentiation of oncolytic virotherapies.
RESUMEN
BACKGROUND: Tumour-associated macrophages (TAMs) are often implicated in cancer progression but can also exert anti-tumour activities. Selective eradication of cancer-promoting (M2-like) TAM subsets is a highly sought-after goal. Here, we have devised a novel strategy to achieve selective TAM depletion, involving the use of T cell engagers to direct endogenous T cell cytotoxicity towards specific M2-like TAMs. To avoid "on-target off-tumour" toxicities, we have explored localising expression of the T cell engagers to the tumour with enadenotucirev (EnAd), an oncolytic adenovirus in Phase I/II clinical trials. METHOD: A panel of bi- and tri-valent T cell engagers (BiTEs/TriTEs) was constructed, recognising CD3ε on T cells and CD206 or folate receptor ß (FRß) on M2-like macrophages. Initial characterisation of BiTE/TriTE activity and specificity was performed with M1- and M2-polarised monocyte-derived macrophages and autologous lymphocytes from healthy human peripheral blood donors. T cell engagers were inserted into the genome of EnAd, and oncolytic activity and BiTE secretion assessed with DLD-1 tumour cells. Clinically-relevant ex vivo models (whole malignant ascites from cancer patients) were employed to assess the efficacies of the free- and virally-encoded T cell engagers. RESULTS: T cells activated by the CD206- and FRß-targeting BiTEs/TriTEs preferentially killed M2- over M1-polarised autologous macrophages, with EC50 values in the nanomolar range. A TriTE with bivalent CD3ε binding - the first of its kind - demonstrated enhanced potency whilst retaining target cell selectivity, whereas a CD28-containing TriTE elicited non-specific T cell activation. In immunosuppressive malignant ascites, both free and EnAd-encoded T cell engagers triggered endogenous T cell activation and IFN-γ production, leading to increased T cell numbers and depletion of CD11b+CD64+ ascites macrophages. Strikingly, surviving macrophages exhibited a general increase in M1 marker expression, suggesting microenvironmental repolarisation towards a pro-inflammatory state. CONCLUSIONS: This study is the first to achieve selective depletion of specific M2-like macrophage subsets, opening the possibility of eradicating cancer-supporting TAMs whilst sparing those with anti-tumour potential. Targeted TAM depletion with T cell engager-armed EnAd offers a powerful therapeutic approach combining direct cancer cell cytotoxicity with reversal of immune suppression.
Asunto(s)
Linfocitos Infiltrantes de Tumor/inmunología , Macrófagos/inmunología , Neoplasias/inmunología , Neoplasias/patología , Subgrupos de Linfocitos T/inmunología , Microambiente Tumoral/inmunología , Adenoviridae/genética , Biomarcadores , Comunicación Celular/inmunología , Línea Celular Tumoral , Citotoxicidad Inmunológica , Expresión Génica , Humanos , Inmunofenotipificación , Activación de Linfocitos/genética , Activación de Linfocitos/inmunología , Linfocitos Infiltrantes de Tumor/metabolismo , Linfocitos Infiltrantes de Tumor/patología , Macrófagos/metabolismo , Macrófagos/patología , Neoplasias/metabolismo , Neoplasias/terapia , Viroterapia Oncolítica , Virus Oncolíticos/genética , Unión Proteica , Subgrupos de Linfocitos T/metabolismo , Subgrupos de Linfocitos T/patología , TransgenesRESUMEN
BACKGROUND: Oncolytic viruses are currently experiencing accelerated development in several laboratories worldwide, with some forty-seven clinical trials currently recruiting. Many oncolytic viruses combine targeted cytotoxicity to cancer cells with a proinflammatory cell lysis. Due to their additional potential to express immunomodulatory transgenes, they are also often known as oncolytic viral vaccines. However, several types of oncolytic viruses are human-specific and the lack of suitable immune-competent animal models complicates biologically relevant evaluation of their vaccine potential. This is a particular challenge for group B adenoviruses, which fail to infect even those immunocompetent animal model systems identified as semi-permissive for type 5 adenovirus. Here, we aim to develop a murine cell line capable of supporting replication of a group B oncolytic adenovirus, enadenotucirev (EnAd), for incorporation into a syngeneic immunocompetent animal model to explore the oncolytic vaccine potential of group B oncolytic viruses. METHODS: Transgenic murine cell lines were infected with EnAd expressing GFP transgene under replication-independent or -dependent promoters. Virus mRNA expression, genome replication, and late protein expression were determined by qRT-PCR, qPCR, and immunoblotting, respectively. We also use Balb/c immune-competent mice to determine the tumourogenicity and infectivity of transgenic murine cell lines. RESULTS: Our results show that a broad range of human carcinoma cells will support EnAd replication, but not murine carcinoma cells. Murine cells can be readily modified to express surface human CD46, one of the receptors for group B adenoviruses, allowing receptor-mediated uptake of EnAd particles into the murine cells and expression of CMV promoter-driven transgenes. Although the early E1A mRNA was expressed in murine cells at levels similar to human cells, adenovirus E2B and Fibre mRNA expression levels were hampered and few virus genomes were produced. Unlike previous reports on group C adenoviruses, trans-complementation of group B adenoviruses by co-infection with mouse adenovirus 1 did not rescue replication. A panel of group B adenoviruses expressing individual mouse adenovirus 1 genes were also unable to rescue EnAd replication. CONCLUSION: Together, these results indicate that there may be major differences in the early stages of replication of group C and B adenoviruses in murine cells, and that the block to the life cycle of B adenoviruses in murine cells occurs in the early stage of virus replication, perhaps reflecting poor activity of Ad11p E1A in murine cells.
Asunto(s)
Adenoviridae/patogenicidad , Proteína Cofactora de Membrana/metabolismo , Viroterapia Oncolítica/métodos , Animales , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos BALB C , Ratones TransgénicosRESUMEN
: Effective immunotherapy of stromal-rich tumors requires simultaneous targeting of cancer cells and immunosuppressive elements of the microenvironment. Here, we modified the oncolytic group B adenovirus enadenotucirev to express a stroma-targeted bispecific T-cell engager (BiTE). This BiTE bound fibroblast activation protein on cancer-associated fibroblasts (CAF) and CD3ε on T cells, leading to potent T-cell activation and fibroblast death. Treatment of fresh clinical biopsies, including malignant ascites and solid prostate cancer tissue, with FAP-BiTE-encoding virus induced activation of tumor-infiltrating PD1+ T cells to kill CAFs. In ascites, this led to depletion of CAF-associated immunosuppressive factors, upregulation of proinflammatory cytokines, and increased gene expression of markers of antigen presentation, T-cell function, and trafficking. M2-like ascites macrophages exhibited a proinflammatory repolarization, indicating spectrum-wide alteration of the tumor microenvironment. With this approach, we have actively killed both cancer cells and tumor fibroblasts, reversing CAF-mediated immunosuppression and yielding a potent single-agent therapeutic that is ready for clinical assessment. SIGNIFICANCE: An engineered oncolytic adenovirus that encodes a bispecific antibody combines direct virolysis with endogenous T-cell activation to attack stromal fibroblasts, providing a multimodal treatment strategy within a single therapeutic agent.
Asunto(s)
Adenoviridae/inmunología , Neoplasias/inmunología , Neoplasias/metabolismo , Virus Oncolíticos/inmunología , Linfocitos T/inmunología , Biopsia , Complejo CD3/metabolismo , Técnicas de Cocultivo , Terapia Combinada , Citocinas/metabolismo , Fibroblastos/metabolismo , Células HEK293 , Humanos , Terapia de Inmunosupresión , Inflamación , Leucocitos Mononucleares/citología , Activación de Linfocitos , Neoplasias/terapiaRESUMEN
Notch signaling plays a fundamental role in determining the outcome of differentiation processes in many tissues. Notch signaling has been implicated in T versus B cell lineage commitment, thymic differentiation, and bone marrow hematopoietic precursor renewal and differentiation. Notch receptors and their ligands are also expressed on the surface of mature lymphocytes and APCs, but the effects of Notch signaling in the peripheral immune system remain poorly defined. The aim of the studies reported here was to investigate the effects of signaling through the Notch receptor using a ligand of the Delta-like family. We show that Notch ligation in the mature immune system markedly decreases responses to transplantation antigens. Constitutive expression of Delta-like 1 on alloantigen-bearing cells renders them nonimmunogenic and able to induce specific unresponsiveness to a challenge with the same alloantigen, even in the form of a cardiac allograft. These effects could be reversed by depletion of CD8+ cells at the time of transplantation. Ligation of Notch on splenic CD8+ cells results in a dramatic decrease in IFN-gamma with a concomitant enhancement of IL-10 production, suggesting that Notch signaling can alter the differentiation potential of CD8+ cells. These data implicate Notch signaling in regulation of peripheral immunity and suggest a novel approach for manipulating deleterious immune responses.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Antígenos de Histocompatibilidad/inmunología , Proteínas de la Membrana/fisiología , Receptores de Superficie Celular/fisiología , Transducción de Señal/fisiología , Animales , Células Presentadoras de Antígenos/fisiología , Apoptosis , Células CHO , Proteínas de Unión al Calcio , Cricetinae , Rechazo de Injerto/prevención & control , Péptidos y Proteínas de Señalización Intercelular , Interferón gamma/biosíntesis , Interleucina-10/biosíntesis , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Endogámicos , Proteínas/fisiología , Receptor Notch2 , Proteínas Serrate-Jagged , TransfecciónRESUMEN
Oncolytic viruses which infect and kill tumour cells can also be genetically modified to express therapeutic genes that augment their anti-cancer activities. Modifying oncolytic viruses to produce effective cancer therapies is challenging as encoding transgenes often attenuates virus activity or prevents systemic delivery in patients due to the risk of off-target expression of transgenes in healthy tissues. To overcome these issues we aimed to generate a readily modifiable virus platform using the oncolytic adenovirus, enadenotucirev. Enadenotucirev replicates in human tumour cells but not cells from healthy tissues and can be delivered intravenously because it is stable in human blood. Here, the enadenotucirev genome was used to generate plasmids into which synthesised transgene cassettes could be directly cloned in a single step reaction. The platform enabled generation of panels of reporter viruses to identify cloning sites and transgene cassette designs where transgene expression could be linked to the virus life cycle. It was demonstrated using these viruses that encoded transgene proteins could be successfully expressed in tumour cells in vitro and tumours in vivo. The expression of transgenes did not impact either the oncolytic activity or selective properties of the virus. The effectiveness of this approach as a drug delivery platform for complex therapeutics was demonstrated by inserting multiple genes in the virus genome to encode full length anti-VEGF antibodies. Functional antibody could be synthesised and secreted from infected tumour cells without impacting the activity of the virus particle in terms of oncolytic potency, manufacturing yields or selectivity for tumour cells. In vivo, viral particles could be efficaciously delivered intravenously to disseminated orthotopic tumours.
Asunto(s)
Adenoviridae/genética , Neoplasias/genética , Neoplasias/terapia , Viroterapia Oncolítica/métodos , Virus Oncolíticos/genética , Transgenes/genética , Adenoviridae/fisiología , Expresión Génica , Genes Reporteros/genética , Neoplasias/virología , Virus Oncolíticos/fisiologíaRESUMEN
Enadenotucirev is an oncolytic group B adenovirus identified by a process of bio-selection for the ability to selectively propagate in and rapidly kill carcinoma cells. It is resistant to inactivation by human blood components, potentially enabling intravenous dosing in patients with metastatic cancer. However, there are no known permissive animal models described for group B adenoviruses that could facilitate a conventional approach to preclinical safety studies. In this manuscript, we describe our tailored preclinical strategy designed to evaluate the key biological properties of enadenotucirev. As enadenotucirev does not replicate in animal cells, a panel of primary human cells was used to evaluate enadenotucirev replication selectivity in vitro, demonstrating that virus genome levels were >100-fold lower in normal cells relative to tumor cells. Acute intravenous tolerability in mice was used to assess virus particle-mediated toxicology and effects on innate immunity. These studies showed that particle toxicity could be ameliorated by dose fractionation, using an initial dose of virus to condition the host such that cytokine responses to subsequent doses were significantly attenuated. This, in turn, supported the initiation of a phase I intravenous clinical trial with a starting dose of 1 × 1010 virus particles given on days 1, 3, and 5.
RESUMEN
BACKGROUND: Enadenotucirev (formerly ColoAd1) is a tumor-selective chimeric adenovirus with demonstrated preclinical activity. This phase 1 Mechanism of Action study assessed intravenous (IV) delivery of enadenotucirev in patients with resectable colorectal cancer (CRC), non-small-cell lung cancer (NSCLC), urothelial cell cancer (UCC), and renal cell cancer (RCC) with a comparator intratumoral (IT) dosed CRC patient cohort. METHODS: Seventeen patients scheduled for primary tumor resection were enrolled. IT injection of enadenotucirev (CRC only) was administered as a single dose (≤ 3 × 1011 viral particles [vp]) on day 1, followed by resection during days 8-15. IV infusion of enadenotucirev was administered by three separate doses (1 × 1012 vp) on days 1, 3, and 5, followed by resection during days 8-15 (CRC) or days 10-25 (NSCLC, UCC, and RCC). Enadenotucirev activity was measured using immunohistochemical staining of nuclear viral hexon and quantitative polymerase chain reaction for viral genomic DNA. RESULTS: Delivery of enadenotucirev was observed in most tumor samples following IV infusion, with little or no demonstrable activity in normal tissue. This virus delivery (by both IV and IT dosing) was accompanied by high local CD8+ cell infiltration in 80% of tested tumor samples, suggesting a potential enadenotucirev-driven immune response. Both methods of enadenotucirev delivery were well tolerated, with no treatment-associated serious adverse events. CONCLUSIONS: This study provides key delivery and feasibility data to support the use of IV infusion of enadenotucirev, or therapeutic transgene-bearing derivatives of it, in clinical trials across a range of epithelial tumors, including the ongoing combination study of enadenotucirev with the checkpoint inhibitor nivolumab. It also provides insights into the potential immune-stimulating properties of enadenotucirev. TRIAL REGISTRATION: This MOA study was a phase 1, multicenter, non-randomized, open-label study to investigate the administration of enadenotucirev in a preoperative setting (ClinicalTrials.gov: NCT02053220).
Asunto(s)
Adenovirus Humanos/fisiología , Carcinoma de Pulmón de Células no Pequeñas/terapia , Carcinoma de Células Renales/terapia , Carcinoma de Células Transicionales/terapia , Neoplasias Colorrectales/terapia , Neoplasias Pulmonares/terapia , Adenovirus Humanos/genética , Administración Intravenosa , Linfocitos T CD8-positivos/metabolismo , Carcinoma de Pulmón de Células no Pequeñas/inmunología , Carcinoma de Células Renales/inmunología , Carcinoma de Células Transicionales/inmunología , Neoplasias Colorrectales/inmunología , Terapia Combinada , ADN Viral/genética , Procedimientos Quirúrgicos del Sistema Digestivo , Humanos , Neoplasias Pulmonares/inmunología , Viroterapia Oncolítica , Virus Oncolíticos/genética , Virus Oncolíticos/fisiología , Procedimientos Quirúrgicos Pulmonares , Resultado del Tratamiento , Procedimientos Quirúrgicos UrológicosRESUMEN
Enadenotucirev (EnAd) is a chimeric group B adenovirus isolated by bioselection from a library of adenovirus serotypes. It replicates selectively in and kills a diverse range of carcinoma cells, shows effective anticancer activity in preclinical systems, and is currently undergoing phase I/II clinical trials. EnAd kills cells more quickly than type 5 adenovirus, and speed of cytotoxicity is dose dependent. The EnAd death pathway does not involve p53, is predominantly caspase independent, and appears to involve a rapid fall in cellular ATP. Infected cells show early loss of membrane integrity; increased exposure of calreticulin; extracellular release of ATP, HSP70, and HMGB1; and influx of calcium. The virus also causes an obvious single membrane blister reminiscent of ischemic cell death by oncosis. In human tumor biopsies maintained in ex vivo culture, EnAd mediated release of pro-inflammatory mediators such as TNF-α, IL-6, and HMGB1. In accordance with this, EnAd-infected tumor cells showed potent stimulation of dendritic cells and CD4+ T cells in a mixed tumor-leukocyte reaction in vitro. Whereas many viruses have evolved for efficient propagation with minimal inflammation, bioselection of EnAd for rapid killing has yielded a virus with a short life cycle that combines potent cytotoxicity with a proinflammatory mechanism of cell death.
RESUMEN
Basic helix-loop-helix (bHLH) transcription factors play a pivotal role in the regulation of tumorigenesis, and also in a wide range of other developmental processes in diverse species from yeast to humans. Here we demonstrate for the first time that Ret finger protein (RFP), a member of the TRIM family of proteins initially identified as a recombined transforming gene from a human lymphoma, is a novel interaction partner for four different bHLH proteins (SCL, E47, MyoD and mASH-1), but does not interact with GATA-1 or PU.1. Interaction with SCL required the B-box and first coiled-coil region of RFP together with the bHLH domain of SCL. RFP was able to repress transcriptional activation by E47, MyoD and mASH-1, but not by members of several other transcription factor families. Transcriptional repression by RFP was trichostatin A sensitive and did not involve an Id-like mechanism or ubiquitination with subsequent degradation of bHLH proteins. Instead, our results suggest that bHLH transcription factors are regulated by a previously undescribed interaction with RFP, which functions to recruit HDAC and/or Polycomb proteins and thus repress target genes of bHLH proteins. These results reveal an unexpected link between the bHLH and TRIM protein families.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas Nucleares/fisiología , Proteínas Represoras/fisiología , Factores de Transcripción/fisiología , Activación Transcripcional/fisiología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Línea Celular , Inmunoprecipitación , Ratones , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína LigasasRESUMEN
BACKGROUND: Notch plays a wide-ranging role in controlling cell fate, differentiation and development. The PI3K-Akt pathway is a similarly conserved signalling pathway which regulates processes such as differentiation, proliferation and survival. Mice with disrupted Notch and PI3K signalling show phenotypic similarities during haematopoietic cell development, suggesting functional interaction between these pathways. RESULTS: We show that cellular responsiveness to Notch signals depends on the activity of the PI3K-Akt pathway in cells as diverse as CHO cells, primary T-cells and hippocampal neurons. Induction of the endogenous PI3K-Akt pathway in CHO cells (by the insulin pathway), in T-cells (via TCR activation) or in neurons (via TrKB activation) potentiates Notch-dependent responses. We propose that the PI3K-Akt pathway exerts its influence on Notch primarily via inhibition of GSK3-beta, a kinase known to phosphorylate and regulate Notch signals. CONCLUSION: The PI3K-Akt pathway acts as a "gain control" for Notch signal responses. Since physiological levels of intracellular Notch are often low, coincidence with PI3K-activation may be crucial for induction of Notch-dependent responses.
Asunto(s)
Fosfatidilinositol 3-Quinasas/metabolismo , Receptores Notch/metabolismo , Transducción de Señal , Animales , Antígenos CD4/metabolismo , Células CHO , Células Cultivadas , Cricetinae , Glucógeno Sintasa Quinasa 3/metabolismo , Humanos , Células Jurkat , Cloruro de Litio/farmacología , Modelos Biológicos , Neuronas/metabolismo , Ratas , Linfocitos T/efectos de los fármacosRESUMEN
Qb bacteriophage virus-like particles (Qb-VLP) are utilized as carriers to enhance immune responses to weakly or non-immunogenic antigens such as peptides and haptens. Qb-VLPs are formed through the self-assembly of multiple Qb capsid protein monomers, a process which traps a large amount of bacterial RNA in the core of the VLP. Bacterial RNA is known to activate the innate immune system via TLR 7 and 8 found within the endosomes of certain immune cells and has been shown to contribute to the immunogenicity of Qb-VLP vaccines. Herein, we evaluated an anti-IgE vaccine comprised of two IgE peptides (Y and P) conjugated to Qb-VLP (Qb-Y and Qb-P, respectively) for in vitro stimulation of human PBMCs and in vivo immunogenicity in mice. The in vitro secretion of IFN-α from human PBMCs exposed to Qb-Y is consistent with TLR7 activation. Immunization of mice with the IgE peptide Qb-VLP conjugates induced high titers of anti-IgE antibodies in wild-type mice, but significantly lower titers in TLR7 knockout mice, supporting the self-adjuvanting role of the RNA. Inclusion of alum and alum/CpG as adjuvants partially or completely compensated for the lack of TLR7 activation in TLR7-deficient mice. Our study demonstrates the key role that TLR7 plays in the immunogenicity of the IgE peptide Qb-VLP conjugate vaccine.
RESUMEN
The anti-human immunoglobulin E (IgE) monoclonal antibody, omalizumab (Xolair®, Genentech, South San Fransisco, CA), is effective in the treatment of poorly controlled moderate to severe allergic asthma and chronic idiopathic urticaria. It acts by specifically binding to the constant domain (Cϵ3) of free human IgE in the blood and interstitial fluid. Although efficacious, use of omalizumab is limited due to restrictions on patient weight and pre-existing IgE levels, and frequent dosing (q2-4 weeks). A vaccine inducing anti-IgE antibodies has the potential for similar clinical benefits with less frequent dosing and relatively lower cost of goods. We developed a vaccine containing two IgE peptide-conjugates targeting the Cϵ3 domain of human IgE. As part of preclinical evaluation of the vaccine to optimize formulation and dose prior to initiating clinical studies, we evaluated the vaccine in non-human primates, and demonstrate the induction of anti-peptide antibodies that can bind to conformationally intact human IgE and are capable, at least in some animals, of substantial lowering circulating IgE levels.
Asunto(s)
Anticuerpos Antiidiotipos/inmunología , Anticuerpos Monoclonales Humanizados , Vacunas Conjugadas/inmunología , Animales , Anticuerpos Monoclonales , Asma/terapia , Humanos , Omalizumab , Primates , Urticaria/terapiaRESUMEN
Since its initial description as a neurogenic locus in Drosophila, the Notch pathway has been shown to play a central role in cell fate decisions across species, including vertebrates, guiding the differentiation of multiple cell types. In the immune system, its function was first demonstrated during lymphopoiesis, but in recent years this pathway has been shown to still be active in peripheral T-cells. Therapeutic opportunities that could arise from the manipulation of Notch signaling in immune disorders such as autoimmunity, allergy and in cancer immunotherapy and transplantation are discussed.
Asunto(s)
Inmunoterapia , Proteínas de la Membrana/fisiología , Linfocitos T/fisiología , Animales , ADN , Humanos , Hipersensibilidad/tratamiento farmacológico , Hipersensibilidad/inmunología , Ligandos , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Proteínas de la Membrana/uso terapéutico , Receptores Notch , Linfocitos T/inmunología , Vacunas/inmunología , Vacunas/uso terapéuticoRESUMEN
Peripheral T cell tolerance is critical in the regulation of immune responses to self antigens, and has implications in the control of autoimmunity, allergic responses and transplant rejection. Here we discuss recent and unpublished data demonstrating that ligation of the cell surface receptor Notch on T cells inhibits immune responses and results in the generation of a regulatory T cell population. Using animal models, we show that prior exposure to antigen can inhibit the response to challenge in an antigen-specific fashion. This inhibition is accompanied by the generation of cells with apparent regulatory capacity and we further show that exposure of T cells to Notch and T cell receptor signals together enhances interleukin 10 production, at least in cell culture systems. We discuss how Notch signalling may integrate with other signals to induce tolerance.